USPTO Patents Application 10687850

(19)

J

Europaisches Patentamt
European Patent Office
Office europeen des brevets

(12)

(11) EP 0 964 059 A2

EUROPEAN PATENT APPLICATION

(43) Date of publication:

15.12.1999 Bulletin 1999/50

(21) Application number: 99304145.8

(22) Date of filing: 27.05.1999

(51) Intel* C12Q 1/00, G01N 27/327

(84) Designated Contracting States:

• Watanabe, Motokazu

AT BE CH CY DE DK ES R FR GB GR IE IT LI LU

Kadoma-shl, Osaka 571-0064 (JP)

MC NL PT SE

• Yoshioka, Toshlhiko

Designated Extension States:

Hirakata-shl, Osaka 573-0035 (JP)

AL LT LV MK RO SI

• Nankal, Shlro

Hirakata-shl, Osaka 573-0071 (JP)

(30) Priority: 11.06.1998 JP 16324398

(71) Applicant: Matsushita Electric Industrial Co., Ltd.

(74) Representative: Price, Paul Anthony King et al

D. Young & Co.,

Kadoma-shi, Osaka-fu, 571-8501 (JP)

21 New Fetter Lane

London EC4A IDA (GB)

(72) Inventors:

• Ikeda, Shin

Katano-shl, Osaka 576-0022 (JP)

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(54) Biosensor

(57) The present invention relates to a biosensor
comprising a working electrode base plate 11 , a counter
electrode base plate 14 and a reagent layer containing
at least an enzyme and an electron mediator, wherein
a working electrode 12 disposed on the working elec-
trode base plate and a counter electrode 15 disposed
on the counter electrode base plate are positioned so
as to mutually face and a terminal of a measuring device
can be brought in contact with terminals 13, 16 of both
electrodes from through-holes 25, 24. Such a biosensor
can produce highly reliable and accurate measuring re-
sults with only a small amount of a sample.

FIG. 3

Printed by Jouve. 75001 PARIS (FR)

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EP 0 964 059 A2

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Description

[0001] The present invention relates to a biosensor for
facilitating a prompt and highly accurate quantitation of.
a substrate contained in a sample.
[0002] Conventionally, polarimetry, colorimetry, re-
ductometry and other methods using various chroma-
tography have been proposed as a method for quanti-
tative analysis of sugars such as sucrose and glucose.
These methods, however, are of poor accuracy, since
their specificity toward sugars is not so high. Of these
methods, polarimetry, whose operation is rather simple,
is greatly influenced by the temperature during the op-
eration, and is not appropriate as a method which ena-
bles ordinary people to make an easy quantitation of
sugars at home.

[0003] Recently, various types of biosensors, utilizing
specific catalysis of enzymes have been developed.
[0004] The following describes quantitation of glu-
cose as an example of quantitating a substrate con-
tained in a sample solution. The method of using glu-
cose oxidase (EC 1.1. 3. 4: hereinafter referred to as
"GOD") and an oxygen electrode or a hydrogen perox-
ide electrode is widely known in the art as electrochem-
ical quantitation of glucose ( for example, 'BIOSEN-
SOR" edited by Shuichi Suzuki, Kodan-sha).
[0005] GOD selectively oxidizes substrate p-D-glu-
cose to D-glucono-5-lactone, using oxygen as an elec-
tron mediator. In the presence of oxygen, oxygen is re-
duced into hydrogen peroxide during this oxidation by
GOD. The decreased amount of oxygen is measured by
the oxygen electrode, or otherwise, and the increased
amount of hydrogen peroxide is measured by the hydro-
gen peroxide electrode. Since both the decreased
amount of oxygen and the increased amount of hydro-
gen peroxide are in proportion to the amount of glucose
contained in a sample solution, the quantitation of glu-
cose can be made from either of these amounts.
[0006] As inferred from the reacting process, this
method has a defect that the results of measurement
are largely affected by the concentration of oxygen con-
tained in the sample solution. Moreover, the measure-
ment becomes impossible in case of the absence of ox-
ygen in the sample solution.

[0O07] Therefore, a new type of glucose sensor sub-
stituting an organic compound such as potassium ferri-
cyanide, a ferrocene derivative or a quinone derivative
or a metal complex for oxygen as an electron mediator
has been developed. With this type of sensor, by oxidiz-
ing a reductant of the electron mediator produced as the
result of the enzyme reaction on the electrode, the con-
centration of glucose contained in the sample solution
can be obtained based on the oxidation current. The
substitution of such an organic compound or a metal
complex for oxygen as an electron mediator makes it
possible to form a reaction layer wherein known
amounts of GOD and the electron mediator therefore
are stably and accurately carried on the electrode. In

this case, since the reaction layer, with its condition al-
most dry, can also be integrated with the electrode sys-
tem, a disposable glucose sensor based on this art has
recently drawn a lot of attention. The typical example of
5 this sensor is the biosensor disclosed in Japanese Pat-
ent Publication No. 2517153. The disposable glucose
sensor facilitates easy measurement of glucose con-
centration by simply introducing a sample solution into
the sensor which is detachably connected to a measur-
10 ing device. This method is applicable to not only quan-
titation of glucose but quantitation of any other substrate
contained in the sample solution.
[0008] In the measurement using above-mentioned
glucose sensor, the concentration of substrate con-
's tained in the sample solution can easily be detected with
a small amount of sample solution (several pi). Howev-
er, a high-performance, easy-to-handle biosensor which
would enable measurement with a much smaller
amount, in particular 1u.l or less of sample solution, is
20 anxiously expected to be developed in various fields in
recent years.

[0009] Also, a conventional electrochemical glucose
sensor comprises an electrode system disposed on sin-
gle plane in most of the cases. If the electrode system
26 is on single plane and an extremely small amount of
sample solution is used, the resistance to charge^trans-
fer between electrodes, mainly ion transfer, is increased
so that a variance in the results of the measurement may
be caused.

30

SUMMARY OF THE INVENTION

[0010] In order to address the above problems, a bi-
osensor in accordance with the present invention com-
as prises a working electrode base plate, a counter elec-
trode base plate and a reagent layer containing at least
an enzyme and an electron mediator, wherein a working
electrode disposed on said working electrode base plate
and a counter electrode disposed on said counter elec-
40 trode base plate are positioned so as to mutually face
having a space therebetween.
[0011] In other words, the working electrode and the
counter electrode are opposed to each other via an
opening space. To form such an opening space, at least
4S one of these base plates may have a curved portion, a
concave portion or the like.

[0012] The present invention provides a biosensor
comprising a working electrode base plate, a counter
electrode base plate, a spacer member placed between
so said both base plates and a reagent layer containing at
least an enzyme and an electron mediator, wherein a
working electrode disposed on said working electrode
base plate and a counter electrode disposed on said
counter electrode base plate are positioned so as to mu-
ss tually face with a spacer member placed therebetween.
[001 3] In this case, it is preferable that at least one of
said working electrode base plate and said counter elec-
trode base plate has a through-hole which exposes an

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electrode terminal of the other plate to outside.
[0014] Namely, when the working electrode base
plate has the through-hole, an electrode terminal of the
counter electrode is exposed to outside. And, when the
counter electrode base plate has the through-hole, an
electrode terminal of the working electrode is exposed
to outside. Of course, both base plates may have the
through-holes.

[0015] It is preferable that one of said working elec-
trode base plate and said counter electrode base plate
has a cut-away portion which exposes an electrode ter-
minal of the base other plate to outside and that a lead
connected to the electrode on a surface of the base plate
having a cut-away portion extends, via a side surface of
the base plate having the cut-away portion, to the back
of the surface where the lead is connected.
[0016] Also, it is preferable that one of said working
electrode base plate and said counter electrode base
plate has a through-hole filled with a conductive material
and a cut-away portion which exposes an electrode ter-
minal of the other base plate to outside and that a lead
connected to the electrode on a surface of the base plate
having the cut-away portion extends, via the conductive
material, to the back of the surface where the lead is
connected.

[0017] The present invention provides a biosensor
comprising an insulating base plate provided with a
groove on its surface, a cover member jointed to said
insulating base plate to form a space for accommodat-
ing a sample in said groove, a working electrode and a
counter electrode disposed so as to mutually face in said
groove and a reagent layer containing at least an en-
zyme and an electron mediator disposed in said groove.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE
DRAWINGS

[001 8] FIG. 1 is an oblique perspective view of a glu-
cose sensor in accordance with one embodiment of the
present invention.

[001 9] FIG. 2 is a broken oblique perspective view of
a glucose sensor in accordance with another embodi-
ment of the present invention.

[0020] FIG. 3 is a broken oblique perspective view of "
a glucose sensor in accordance with still another em-
bodiment of the present invention.
[0021] FIG. 4 is a broken oblique perspective view of
a glucose sensor in accordance with another embodi-
ment of the present invention.

[0022] FIG. 5 is a broken oblique perspective view of
a glucose sensor in accordance with still another em-
bodiment of the present invention.
[0023] FIG. 6 is a broken oblique perspective view of
a glucose sensor in accordance with another embodi-
ment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED
EMBODIMENTS

[0024] In the following, the present invention is de-
5 scribed in detail referring to embodiments.

Embodiment 1

[0025] As an example of biosensor, glucose sensor is
10 explained.

[0026] Fig. 1 describes the appearance of a glucose
sensor in accordance with one embodiment of the
present invention.

[0027] A working electrode base plate 1 was made as
15 follows. Palladium was sputtered on an insulating base
plate to form a working electrode and a leadAerminal
portion. Then, an insulating member 7 was attached to
the base plate to form a working electrode 2 and a ter-
minal 3 which was inserted into a measuring device.
20 [0028] Meanwhile, by using an insulating base plate
with a curved portion 6 which was bloated toward out-
side, palladium was sputtered on the internal concave
wall of said curved portion 6 to form a counter electrode
5. This was how a counter electrode base plate 4 was
25 formed. The end of the curved portion was provided with
an air vent 8.

[0029] An aqueous solution containing GOD and po-
tassium ferricyanide which was an electron mediator
was dropped on the working electrode 2 on the working
30 electrode base plate 1 , then dried to form a reagent lay-
er.

[0030] Lastly, the working electrode base plate 1 and
the counter electrode base plate 4 were laminated to
produce a glucose sensor. By this lamination, the work-

35 ing electrode 2 and the counter electrode 5 were dis-
posed so as to mutually face having a space between
the working electrode base plate 1 and the curved por-
tion 6. This space accommodated a sample, and if a
sample solution was brought in contact with the open

40 end of this space, a capillary phenomenon moved the
sample solution toward the air vent to reach the elec-
trode system.

[0031] An aqueous solution containing a predeter-
mined amount of glucose was supplied as a . sample to

45 the space of the sensor. After a predetermined time, a
voltage of 500mV was applied to the working electrode
2 using the counter electrode 5 as reference. As for the
counter electrode 5, electrical conductivity was obtained
by fastening the end of the curved portion 6 with a clip,

50 for example. When the value of the current which flowed
between the working electrode and the counter elec-
trode by this voltage application was measured, the cur-
rent response in proportion to the glucose concentration
in the solution was observed. Glucose reacted with fer-

55 ricyanide ion and GOD in the solution and, as the result,
glucose was oxidized to glucono lactone while reducing
ferricyanide ions to ferrocyanide ions. The concentra-
tion of this ferrocyanide was in proportion to the glucose

EP 0 964 059 A2

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concentration. Thus, the glucose concentration could be
measured based on the oxidation current.
[0032] Compared to the case where almost equal
amount of a sample was introduced to the sensor com-
prising an electrode system disposed on single plane of
a base plate, an increase in the response value was ob-
served in this embodiment This is presumed because
the electrode system was disposed so as to mutually
face so that ion transfer between the electrodes is facil-
itated.

Embodiment 2

[0033] Fig 2 is a broken oblique perspective view of a
glucose sensor in accordance with the embodiment of
the present invention with an omission of a reagent lay-
er, and shows one example of the arrangement of elec-
trode/lead.

[0034] A working electrode base plate 11 was made
as follows. First, a silver paste was screen-printed on
an insulating base plate of polyethylene terephthalate
to form a lead 1 3. Then, a conductive carbon paste con-
taining a resin binder was printed on the base plate to
form a working electrode 12, which was in contact wrth
the lead 1 3. Subsequently, an insulating paste was print-
ed on the base plate 11 to form an insulating layer 17,
which covered the peripheral portion of the working
electrode 1 2 so as to hold the exposed area of the work-
ing electrode 1 2 constant.

[0035] In the same manner as described above, a
counter electrode base plate 14 was made. Specifically,
after a silver paste was printed on the backside of an
insulating base plate toform a lead 1 6, a conductive car-
bon paste was printed to form a counter electrode 15
and an insulating paste was printedtoform an insulating
layer 18. Thecounter electrode base plate was provided
with an air vent 19.

[0036] A spacer 21 placed between the working elec-
trode base plate 1 1 and the counter electrode base plate
14 had provided with a slit 22, which formed a sample
solution supply pathway between the working electrode
base plate and the counter electrode base plate.
[0037] In the same manner as the embodiment 1 , a
reagent layer was formed on the working electrode base
plate. Then, the working electrode base plate 11, the
counter electrode base plate 14 and the spacer 21 were
adhered to each other in a positional relationship as
shown by the dotted line in Fig. 2 to produced biosensor.
The counter electrode and the working electrode having
the reagent layer thus faced each other in the sample
solution supply pathway formed at the slit 22 of the spac-
er 21 The air vent 1 9 of the counter electrode base plate
was connected to this sample solution supply pathway,
Thus if a sample solution was brought in contact wrth a
sample solution supply inlet 23 formed on the open end
of the slit, a capillary phenomenon caused the sample
solution to reach the reagent layer in the sample solution
supply pathway.

[0038] Then, glucose was measured in the same
manner as the embodiment 1 .
[0039] The strength of sensor against physical pres-
sure applied to the base plate was increased by the
5 placement of the spacer between the both base plates.
Thus the volume of the sample solution supply pathway
was easily kept constant, so that the influence of phys-
ical pressure and the like on the sensor response was
diminished.

10 [0040] As the result of measurement, a current re-
sponse in proportion to the glucose concentration con-
tained in the solution was observed and a variance in
the response was reduced.

15 Embodiment 3

[0041] Fig. 3 is a broken oblique perspective view of
a glucose sensor in accordance with the embodiment of
the present invention with an omission of a reagent lay-

[0042] This sensor had the same configuration as the
embodiment 2 except that the working electrode base
plate 1 1 and the counter electrode base plate 1 4 respec-
tively had a through-hole 24 and 25 for exposing a ter-
25 minal to outside.

[0043] By the provision of a through-hole to each ot
both base plates, a part of leadAerminal 16 of the coun-
ter electrode base plate 1 4 was exposed to outside from
the through-hole 24 of the working electrode base plate
30 11 while a part of lead/terminal 1 3 of the working elec-
trode base plate 11 was exposed to outside from the
through-hole 25 of the counter electrode base plate 14.
If the spacer 21 would horizontally extend to the termi-
nals, the spacer might be provided with a corresponding

35 through-hole.

[0044] This provision of the through-hole secured the

fitting of a laminating-type sensor chip into the measur-
ing device, i.e., the electrical connection of the sensor
chip and the measuring device, which lead to the im-
40 provement in the measuring accuracy.

Embodiment 4

[00451 Fig 4 is a broken oblique perspective view of
45 a glucose sensor in accordance with the embodiment of
the present invention with an omission of a reagent lay-
er

[0046] A working electrode base plate 11 and a spac-
er 21 had the same configuration of the embodiment 2.
50 [00471 On the other hand, a counter electrode base
plate 34 was formed as follows. Palladium was sput->
tered on the whole surfaces (including the sides) of an
insulating base plate provided with a cut-away portion
36 which was formed by cutting away the portion cor-
55 responding to a terminal 13 ot the working electrode
base plate 11 . A palladium layer thus formed under the
counter electrode base plate 34 functioned as a counter
electrode, which was electrically connected to the ter-

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EP 0 964 059 A2

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minal of the palladium layer formed on the side and the
upper surface of the base plate.
[0048] In the same manner as the embodiment 1 , a
reagent layer was formed on the working electrode of
the working electrode base plate. Then, the working
electrode base plate 11, the counter electrode base
plate 34 having an air vent 39 and the spacer 21 were
adhered to each other in a positional relationship as
shown by the dotted line in Fig. 4, to produce a biosen-
sor.

[0049] By the provision of the cut-away portion 36 to
the counter electrode base plate 34, a part of lead/ter-
minal of the working electrode base plate 11 was ex-
posed to outside from the cut-away portion 36. If the
spacer 21 would horizontally extend to the position cor-
responding to the terminals, the spacer 21 might be pro-
vided with a corresponding cut-away portion. Mean-
while, the lead electrically connected to the counter
electrode 35 extended via the side of the counter elec-
trode base plate 34 to the upper surface thereof.
[O0S0] This enabled both terminals to be exposed to
outside of only one base plate. Therefore, the conven-
tional connecting terminal of the measuring device wide-
ly used could be applied to the sensors of above-men-
tioned configuration without making any changes ther-
eon, which was effective for reducing the manufacturing
cost of sensors.

[0051] A lead disposed on a side of a sheet-like base
plate might have a problem of physical strength, com-
pared to a lead disposed on a upper surface of the plate
or under the plate. In such a case, as shown in Fig. 5, it
was also possible that the counter electrode base plate
34 might be provided with a through-hole 37 filled with
a conductive material such as a silver paste, a carbon
paste or the like. Then, the lead of the electrode dis-
posed under the base plate might be connected via this
conductive material to the terminal on the base plate.
[0052] In this embodiment, the counter electrode
base plate 34 was provided with the cut-away portion
36 or the through-hole 37, and, even if the working elec-
trode base plate 11 might be provided with these cut-
away portion and through-hole, the same effects could
be obtained. In this case, it would be required to deter-
mine the area of the counter electrode using an insulat-
ing layer and so forth.

Embodiment 5

[0053] Fig. 6 is a broken oblique perspective view of
glucose sensor in accordance with the embodiment of
the present invention with an omission of a reagent lay-
er.

[0054] An insulating base plate 40 was provided with
a groove 41 whose outer surface and upper surface
were open. Palladium was sputtered on the side walls
facing each other of the groove 41 and a upper surface
of the insulating base plate. And the base plate was
trimmed by laser to form a working electrode 42, a coun-

ter electrode 45 and leadAerminal portions 43 and 46
corresponding with each electrode. Also, an insulating
layer 47 was formed so as to partially cover the said
lead. Next, an aqueous solution containing GOD and

5 potassium ferricyanide was dropped on the groove 41
and dried to form a reagent layer. Thereafter, a cover 48
provided with an air vent 49 at a position corresponding
to the innermost of the groove was adhered to the base
plate 40 in a positional relationship as shown by the dot-

io ted line in Fig. 6 to produce a biosensor.

[0055] In this biosensor, the groove 41 of the base
plate was the place where a sample was accommodat-
ed, and if a sample solution was brought in contact with
the open end of the groove 41 on the end of the base

15 plate, a capillary phenomenon moved the sample solu-
tion toward the air vent to reach both electrodes.
[0056] As for the sensors such as above-mentioned
embodiments wherein both base plates having an elec-
trode were laminated, a discrepancy in position of the

20 base plates might occur in their laminating process.
However, as for the sensor of this embodiment wherein
electrode systems were formed on the internal wall of
the groove 41, such discrepancy induced by laminating
process did not occur, thereby not causing a decline of

25 measuring accuracy.

[0057] The voltage applied to the electrode system in
the above-mentioned embodiments was 500mV, but
might not be limited thereto if the voltage might cause
the oxidization of the electron mediator which is reduced

30 in the enzyme reaction.

[0058] As the oxidoreductase contained in the reac-
tion layer, the one corresponding to a substrate to be
analyzed contained in the sample solution can be used.
For this purpose, fructose dehydrogenase, glucose ox-

35 idase, alcohol oxidase, lactic acid oxidase, cholesterol
oxidase, xanthine osidase and amino acid oxidase are
exemplified.

[0059] As the electron mediator, potassium ferricya-
nide, p-benzoquinone, phenazine methosulfate, meth- .
40 yien blue and ferrocene derivatives are exemplified. Al-
so, oxygen can be used as the electron mediator to ob-
tain current response. One or more of these electron
mediators are used.

[0060] The enzyme or the electron mediator may not
45 be dissolved in the sample solution when the reagent
layer is fixed to the base plate. In case it is fixed, Cross-
linking method or Absorption method is preferable. They
may be blended into electrode materials.
[0061] In the above embodiments, the through-hole
50 and the cut-away portion provided to the base plate
were described in order to bring a specific electrode sys-
tem, lead/terminal and the connecting terminal of the
measuring device in contact with said terminal, but their
shape, arrangement and so forth are not limited to those
55 described in the embodiments.

[0062] Also, in the above embodiments, carbon and
palladium are used as electrode materials, but electrode
materials are not limited to those. As working electrode

5

1

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EP 0 964 059 A2

materials, any conductive materials can be used so lonn

dizahon of the electron mediator. As counter electrode
magnate, any conductive materials such as ster or
pUram which is generally used can be used
KL h S PreSen ' inVen ' i0n ' as menli °"ed above.
5? ac Lra^mT Mn Pr0dUCe «
aln ^ , L7.e aSUnn9 reSU " S '* 3 Sma »

10

Claims

1. A biosensor comprising a working electrode base
Plate, a counter electrode base plate and a reagent
teye conta.mnganeastanenzymeanda neleclron
3? ■!*•*' a working electrode disposed™

? eiec,rode base p,ate * «

electrode disposed on said counter electrode base
Plate are positioned so as to mutually face with a
space between the electrodes.

10

is

20

2.

A brosensor comprising a working electrode base
Plate a counter electrode base plate, a spacer

a2l^ be,WMnffiil,b0,hbase P bt ^ *'
a reagent layer containing at least an enzyme and

an electron mediator, wherein a working eLtrode
drsposed on sa,d working electrode base plate and
acoumer electrode disposed on said countered

Strops 3 SP8Cer member P,aC8d b9,W9en ,h °

3. The biosensor in accordance with claim 1 or 2

SHI^f T °' WOrkin9 e,ec,rode ba <* 35
Plate and sad counter electrode base plate has a

through^ole wh«h exposes an e.ectrooe JSi

of the other plate to outside.

». ^ biosensor in accordance with claim 1 or 2 40
wherein one of said working electrode base plate
and sa,d counter electrode base plate has a cut-

rhe P r° n h WhiCh , eXP0SeS an e,ec,rode 'ermtna!

*1 * Pte ' 9 t0 ou,side ** a lead con-
nected to the electrode on a surface ol the base «

t„,fA . ,h f baSepb,ehavin 9 ac "t-awayporti OT
tothebackof the surface where the lead isconne^

The biosensor in accordance with claim 1 or 2
wherem one of said working electrode base plate
and sa,d counter electrode base plate has a
hroug h . ho(e fl|led ^ a ^ las a

nal of the o her plate to outside and a lead connect-
ed to the electrode on a surface of the base plate
hav,ngacut-away portion extends, via saidconduc

Jive .material, to the back of the surface where the
lead is connected.

^ biosensor comprising an insulating plate provided
w,.h^rooveoni.s S urface.acovermemberjoine1
to sa,d .nsu.at.ng base pia.e to form a space for ac-
commodating a sample in said groove a workino
electrode and a counter electrode disposes
to mutually face in said groove and a reagent layer
coming at leas, an enzyme and an electron Z
diator disposed in said groove.

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FIG. 1

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FIG. 2

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FIG. A

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FIG. 5

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FIG. 6

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